The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages . The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.Arachidonic acid (ARA, 20:4 These may regulate and impact immune function and inflammatory responses, exerting both pro-inflammatory and anti-inflammatory effects (n-6) and dihomo- γ-linolenic acid  are precursors for different types of bioactive eicosanoids. Cyclooxygenases (COX) initiate the conversion of ARA and DGLA to prostaglandins, among them prostaglandin series 1 and series 2 , respectively (Lobosphaera incisa wild type (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively  groups are known to affect a wide range of physiological processes , 2. In aectively . Primariectively , 5. Howeectively , 8. Receectively –16. In tectively –21. Howeectively , 23, whoenomenon . Microalenomenon . Aquaculenomenon . MicroalStreptococcus iniae. Furthermore, fish surviving the S. iniae challenge were analyzed to understand the role of modified diets in modulating immune gene expression and FA composition during infection. Selected doses of microalgae for dietary supplementation were based on previous studies that were conducted in our laboratory, showing positive results at supplementation rates similar to those selected in this study  composition, expression of immune and inflammatory genes, and resistance to infection with is study , 11, 28.Danio rerio) were raised in the lab and maintained at a temperature of 28°C with a light:dark cycle of 12:12 h. Prior to the start of the trial, fish were fed at 2% of their body weight once a day with a commercial feed . During the entire experimental period, tanks were siphoned every other day, and 10% of water exchange was carried out. Temperature was monitored daily, and measurements of water quality parameters including ammonia and nitrite were monitored twice a week using colorimetric test kits . Ammonia (0–0.2 mgL−1), nitrite (0–0.025 mg L−1), nitrate (0–10 mg L−1), and oxygen were maintained at over 80% saturation. The experimental trial was approved by the Ben-Gurion University Committee for the Ethical Care and Use of Animals in Experiments (authorization no. IL 58-09-2016).Wild type zebrafish  by an intraperitoneal (IP) injection of 10 μl, using a high precision syringe pump-Fusion 720 . Mortality was monitored daily for a period of 1 week. Fish were sampled at the completion of 4 weeks of dietary supplementation for determining FA composition, PGE2 production and gene expression analysis. For each experimental treatment, 20 fish were sampled for gene expression analysis (liver and kidneys), and another 20 fish were sampled for determining FA composition and prostaglandin production. Organs were weighed at sampling using analytical scales. Each sample consisted of a pool of five fish from every tank, with four pools per treatment. All sampled fish were euthanized in clove oil (250 ppm) prior to dissection. Liver and kidney samples collected for gene expression analysis were immediately placed in RNA later™ (100 mg/500 μl) at room temperature and left for 6 h at 4°C before transferring to −80°C for storage. Liver and kidney samples to be analyzed for FA composition and prostaglandin production, respectively, were collected in 1.5-ml tubes on dry ice, and immediately snap-frozen in liquid nitrogen and stored at −80°C until analysis. A similar method of sampling and sample processing for gene expression, FA composition and PGE2 production was repeated for surviving fish after completing 1 week of the S. inae challenge.The dietary trial was designed to test five different treatments comprising two supplementation levels of two types of dried broken algae and, consequently, of ARA and DGLA, and one group of an unsupplemented control. Similarly sized adult zebrafish (ca. 5–6 months old), originating from a single cohort, were distributed among 20 aquaria, 30-L each (30 fish/tank), supplied with individual submerged biological filters and aeration. Each of the five treatments was tested in four replicates. Fish were fed with experimental and control diets for a period of 4 weeks at 2% body weight per day, in two separate feed applications. Fish weight was determined at the beginning of the trial  and then again after the 4 weeks of feeding, by weighing 20 fish from each treatment group. At the end of this period, 20 fish from each aquarium were infected with S. iniae in a 103 cfu ml−1 cell suspension. This dose was pre-calibrated and confirmed to induce 50% mortality in the zebrafish . The inoculum was prepared by inoculating frozen bacterial stock (from −80°C) into fresh TSB broth in a 15-ml tube. The cultures were grown at 25°C to the mid-logarithmic phase of growth . One milliliter of this culture was centrifuged at 1500 × g for 10 min at 4°C and washed in sterile phosphate buffered saline (PBS). Serial dilutions were carried out from this tube to achieve a final inoculum concentration of 103 cell ml −1. The final cell concentration was confirmed by subsequent plating of the inoculum on TSB plates using the drop plate method.After 1 month of dietary supplementation, the fish were challenged by an IP injection of a 10-μl inoculum of EF1 was chosen as the internal control after confirming its stable expression within all treatments (EF1α using the formula 2(−ΔCt), where ΔCt = avg. of target gene Ct—avg. of EF1 gene Ct.Frozen tissue samples were homogenized in a mixer mill  using metal beads with continuous cooling using liquid nitrogen. RNA extraction from tissue homogenates was carried out using a SV Total RNA Isolation Kit  based on the manufacturer's instructions. Total RNA was eluted and quantified on a Nanodrop spectrophotometer  and reverse transcribed using the Verso cDNA Synthesis Kit  following the manufacturer's instructions. The resulting cDNA was used as a template for quantitative PCR in a Biorad CFX96™ real-time PCR detection system with SsoAdvanced™ Universal SYBR Green Supermix . Primers used in this study are shown in Table eatments . A melt 2 flow. Separation of total lipid extracts into neutral and polar lipid fractions was performed by sequential elution with chloroform and methanol on silica gel cartridges . Polar and neutral fractions were then transmethylated, and fatty acid methyl esters (FAMEs) were extracted as described in Nayak et al. , to avoid sample oxidation. The columns were washed by ice-cold water, 15% ethanol and hexane, and the PGE2-containing fraction was eluted in ethyl acetate. The samples were dried under reduced oxygen conditions and re-dissolved in 500 μl of assay buffer. PGE2 was quantified by a PGE2 EIA kit .Kidney samples were solubilized in 500 μl of ice cold acidified DDW (pH = 3.5), mixed vigorously, and homogenized on ice. The supernatant was collected and 100 μl aliquots were mounted on C18 flash columns , under reduced oxygen conditions  and fixed in 10% neutral buffered formalin. Fixed whole fish were cut into ca. 0.4-cm-thick transverse sections and processed using routine histological techniques as described in Sharon et al. . The slipost hoc test . The significance between gene expression values before and after challenge was determined using the t-test. Survival curves were generated by the Kaplan Meyers Survival Analysis and treatments compared using the Holm-Sidak method (Sigma Plot 13). A principal component analysis (PCA) was carried out using the FactoMineR package, and plots for the PCA were generated using ggplot 2 package in R studio version 1.14.The results of gene expression, relative FA compositions and histological analyses between experimental groups were compared by a one-way analysis of variance (ANOVA), using the Sigma Plot 13 software . Pairwise multiple comparisons were carried out using the Dunn's L. inicisa WT and MUT significantly increased the content of ARA and DGLA in the supplemented diets, respectively, as compared to the control. Feed supplemented with 7.5 and 15% of WT contained elevated ARA levels, of 11.6 and 21.8 μg mg−1, respectively, compared to 0.6 μg mg−1 in the control. The amount of DGLA in these diets was similar to the level in the control, 0.3 and 0.6 μg mg−1 feed, respectively . Total fatty acid content in the control feed was lower (76.1 μg mg−1) than in the supplemented feeds, in which levels ranged from 96–116.5 μg mg−1, indicating an increased fraction of microalgal FA. In line with the higher proportion of oleic acid  in P127 biomass , compared to the WT-supplemented and control feeds (15.7–16.3 % of TFA). In the WT-supplemented diets, the marked increase was solely in ARA levels, due to elevated ARA levels in the WT  and docosahexaenoic acid , in the experimental diets by up to 42% as compared to the control. It is worth noting that the supplementation did not affect the protein and carbon content in the diets , and linoleic acid , accounting for about 65% of neutral lipid and about 44% of polar lipid fatty acids. Among the highly unsaturated fatty acids (ω3 HUFA), DHA was present at a higher proportion in the polar lipid fraction in all treatment groups (ranging from 16.3 to 19.6% of TFA) as compared to DHA levels in the neutral lipids. The proportion of EPA in both polar and neutral fractions was <5% of TFA. Dietary supplementation with WT and MUT resulted in a significant increase in the proportion of ARA and DGLA, as a % of TFA, in both the polar and neutral lipid fractions of fish liver as compared to the control  was observed in the liver of zebrafish, corresponding to the dietary supplementary levels , oleic acid  higher as compared to the control .To examine the effect of dietary manipulations on disease resistance, fish were challenged with TLR 22, a membrane receptor activated by pathogen-associated molecular patterns (PAMPs) and the pro-inflammatory cytokine TNF-α, were significantly downregulated in fish fed with both supplemented diets, as compared to the control. The gene expression of another pro-inflammatory cytokine, interleukin 1β, did not change between treatment groups  was moderately elevated only in the MUT 15% supplemented group, whereas the gene expression of LOX-2  was downregulated in the WT-supplemented group , were downregulated in the liver of fish fed with the experimental diets, although differences were significant only for the phospholipase C-γ1 gene  and phospholipase A2 . The overall expression of TNFα was significantly higher in fish fed with WT than in fish fed with MUT and the control diets  and GPX were affected by the 15% MUT- supplemented diets, whereas genes such as TNFα, TL22, PLA2, and PLCγ were affected by the control diet.A principal component analysis (PCA) was performed on the normalized expression values obtained from the qPCR, to visualize patterns of gene expression and its modulation under different dietary treatments. Data are displayed in a scatter plot view with different colors representing the dietary treatment Figure . The bion-6 LC-PUFA's pro- and anti-inflamatory effects may not be that apparent. Furthermore, ARA metabolism also generates resolving molecules, such as lipoxin A4, essential for inflammation resolution. The n-6 LC-PUFA-associated metabolic pathways that lead to the distinct immunomodulatory effects involve eicosanoid signaling, which modulates a range of physiological conditions related to immune response and inflammation. Omega-6 LC-PUFA and their derivatives are capable of modifying cell membrane composition and the regulation of target transcription factors and host gene expression  was replaced with broken biomasses of two strains of the oleagenous microalga arvation , 37. Henpplement . It is iAs a major hematopoietic organ, the kidneys contain large numbers of mature, active leucocytes  and weren-6-PUFA and their metabolites are well known to affect gene expression by activating nuclear factors such as NF-κB  exert an effect on NF-κB expression similar to ARA. In the present study, there was no evident increase in PGE2 following dietary supplementation with any of the experimental diets. The lack of increase in PGE2 is in line with the lack of changes in COX 1 and 2 gene expression, which are enzymes that produce prostaglandins from ARA and DGLA. In sharp contrast to the upregulation of NF-κB, all the other inflammation-related genes, the cytokines TNFα and IL-1ß and the membrane-receptor TLR-22, were either downregulated or unaffected by the supplemented diets. This clearly suggests that the expression of NF-κB was not as a result of the canonical TNF activation pathways, as cytokines were suppressed with the supplemented diets. This important finding may indicate the beneficial effect of microalgae-supplemented feeding following the bacterial challenge. Although the exact mechanisms for activation of NF-κB by ARA- and DGLA-rich microalgae remains unclear, an important factor that may induce its expression could be the increase in ROS and oxidative stress due to highly PUFA-rich diets  was observed. These are in line with the unchanged PGE2 levels prior to infection, as determined by EIA. However, an increase in LOX1 expression, which is homologous to 15-LOX in humans, was noticed in the kidneys of 15% MUT-fed fish. This could be indicative of the enhanced conversion of DGLA to 15-(S)-hydroxy-8, 11, 13-eicosatrienic acid (15-HETrE), which is catalyzed by 15-LOX. The 15-HETrE effects are reported to be associated with suppression of chronic inflammation, vasodilation, lowering of blood pressure, arresting of cancer cell growth and differentiation of tumor cells  was evident in the WT- and MUT-supplemented groups. Phospholipase A2 is mostly responsible for releasing ARA from membrane phospholipids. Similarly, . The release of DAG and IP is responsible in the downstream events that lead to the activation of transcription factors and cytokines (n-3-LC-PUFAs has been reported in lymphocytes (n-6-LC-PUFA-rich feeds. A higher expression of PLCγ has been reported in cancer cells where it acts as a signaling intermediate for cytokines such as interleukins, and it was also reported to promote tumorgenesis through intracellular and extracellular signaling pathways (n-6 LC-PUFA ARA and DGLA, and correlates with the reduced expression of pro-inflammatory TLR22 and TNFα.In this study, supplemented diets altered the expression of genes encoding enzymes involved in arachidonic acid metabolism. No significant change in the expression of the cyclooxygenase genes (or cells . The enhrom DGLA , 48. Themilarly,  reportedytokines , 51. Difytokines . A similphocytes , 53; howpathways , 55. Ove2O2 generated from the dismutation of the superoxide radical (S. iniae challenge.The 15% MUT-supplemented diet resulted in elevated gene expression of catalase and glutathione peroxidase pre-challenge. Both catalase and glutathione peroxidase remove the Hn of O2- . Expressn of O2- . InteresS. iniae challenge suggest that the immune modulation induced by experimental diets effectively protected the fish from infection, although it is evident that the pre-challenge immune modulation induced by the treatments differed between the WT- and MUT-supplemented groups. The gene expression analysis occurring 1 week after challenge, when mortalities subsided, represents the period of recovery from infection. The elevated lysozyme expression, at the recovery from infection stage in the WT-supplemented fish, was in line with the elevated expression of the COX-2 gene, which is generally associated with a pro-inflammatory effect. Changes in gene expression of TNF-α, TLR-22, and NF-κB in the control were in line with the pre-challenge levels in the treatment groups, which were better protected from the infection. It is difficult to point which of the genes could directly explain the higher survival in the treatment groups. It is likely that a combined effect of the various immune factors produced the final protective outcome.The mortality results following the COX and LOX genes in the surviving fish. Remarkably, the PGE2 levels were significantly elevated in fish from all treatment groups following the S. iniae challenge. Bacterial lipossacharides are a known stimulant for PGE2 synthesis (2 levels, an impact on other eicosanoids, in particular on PGE1, in MUT-supplemented diets should not be ruled out. PGE2 is generally regarded as a pro-inflammatory mediator that facilitates the formation of inflammatory cytokines (2, which was shown to downregulate 5-LOX and upregulate 15-LOX, promoting the formation of anti-inflammatory lipoxins (2 may exhibit both pro-inflammatory and anti-inflammatory actions. The expression of COX-2 and the subsequent elevation in PGE2 production were documented to be associated with bacterial infections (2 levels was expected, though surprisingly, there was no apparent treatment effect. Only in the 15% WT-supplemented group, which provided the highest ARA, was a substantial increase in COX-2 gene expression measured in the same groups and correlated with the significant increase in PGE2. Cyclooxygenases are known to have a higher affinity to ARA as compared to DGLA, so higher PGE2 levels were expected in the ARA-supplemented groups, yet levels were similar among all treatment groups. It should be noted that the PGE2 assay kit used in this study exhibits up to 70% cross-binding with PGE1, which is the product of COX enzyme activity on DGLA. Thus, it is possible that the similar levels obtained were due to cross-reactivity. Since the post-challenge ARA levels decreased in all treatment groups, it can be assumed that this fatty acid was metabolized, including by COX enzymes, to substances that aided in the control of bacterial infection.Our results showed significant change in the expression of the ynthesis , 59, whiynthesis , 61. Thoytokines . Recent lipoxins , 64. Thufections , 65, 66.LOX-1 and LOX-2 genes, during the post-infection recovery period in the treatment groups, as compared to the control, is striking. LOX-1 and LOX-2 are homologous to the human 15-LOX and 5-LOX. 15-LOX is involved in the generation of the pro-resolving lipoxin A4 from ARA, suggesting that the higher expression aids in resolving the infection (The elevated expression of the nfection . LOX 15 nfection ; thus, infection . Previounfection –72. The nfection . These mnfection . As emphnfection , it is eS. inaie challenge appeared to upregulate expression of IL1β in all treatments, but most prominently in the WT-supplemented diets. The upregulation of IL1β was expected as it is a cytokine mediating the inflammatory response toward endotoxin and liposaccharides (L. incisa (L. incisa residue supplemented through artemia enrichment (The charides , 76. Thecharides , 78. The. incisa . Similarrichment .n-6 LC-PUFA, WT, containing ARA, and the MUT, containing DGLA. Microalgae are a rich resource of functional food ingredients, encompassing health-promoting carotenoids, antioxidants, vitamins, essential aminoacids, and immunomodulating polysaccharides. Though we focused on the effects related to n-6 LC-PUFA application, more investigations should be performed in order to better understand the consequences of L. incisa-supplementation on the enhanced resistance to bacterial infection and immunomodulation in zebrafish. An important outcome of this work is that not only the balance between n-3 and n-6 LC-PUFA is important in sustaining the inflammatory homeostasis, but elevation in the dietary provision of n-6 LC-PUFA via microalgae-enriched diet exerts an apparent immunomodulatory effect in zebrafish and thus can be suggested as a potential health-promoting fish feed supplement.In this work, we focused on the potent effects of major n-6 LC-PUFA in experimental microalgae-supplemented diets, which appear to substantially affect immune function and infection resistance. It should be noted that microalgal biomass provides additional components that likely affected the immune and health status of treated fish, yet since the same alga was used in both cases, the main difference was in the DZ, IK-G, and SN planned and carried out the experiments and the resulting interpretation. The EIA assay was carried out by GC. All authors contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Studies have shown that perioperative inflammatory response is one of the important factors that caused postoperative cognitive dysfunction (POCD). Ulinastatin is a broad-spectrum protease inhibitor that inhibits inflammatory. We investigated the effects of ulinastatin on inflammatory response and early postoperative cognitive function in elderly patients undergoing spinal surgery.Methods: This clinical trial was approved by the Xuanwu Hospital Ethical Committee (Registration number: ChiCTR-IPR-16008931). Sixty elderly patients undergoing elective spinal surgery with American Society of Anesthesiologists (ASA) status of I–II were randomized into ulinastatin and control groups; total intravenous anesthesia was performed. The elderly patients in ulinastatin group underwent intravenous infusion of ulinastatin 10,000 units/kg following anesthesia induction and before surgical incision, and 5000 units/kg on post-operative days 1 and 2. Cognitive function was determined with Montreal Cognitive Assessment (MOCA) test preoperatively and on post-operative day 7 by a neurologist. Serum lipopolysaccharide (LPS), interleukin-6 (IL-6), C-reactive protein (CRP), and matrix metalloprotease-9 (MMP-9) concentration levels were measured at baseline, the end of surgery, and on post-operative days 1 and 3.Results: All elderly patients completed the study. Ulinastatin infusion significantly reduced the incidence of POCD in elderly patients undergoing spine surgery . The elderly patients in ulinastatin group exhibited lower serum LPS, IL-6, CRP, and MMP-9 concentrations, as well as a shortened peak value duration, compared with those in the control group following surgery (P < 0.05).Conclusion: Systemic inflammation and translocation of LPS were inhibited by the infusion of ulinastatin in elderly patients undergoing spinal surgery. The anti-inflammation intervention with ulinastatin can significantly improve the elderly patients’ postoperative cognitive function. The mechanism of postoperative cognitive dysfunction (POCD) in elderly individuals is related to excessive systemic inflammation caused by surgical trauma in elderly individuals, as well as an imbalance in the circulatory and central nervous system’s immune responses (increased pro-inflammatory substances and reduced anti-inflammatory substances) . PeriopeOrganic inflammatory response can promote the generation of MMP-9. MMP-9 can resolve and destroy the basement membrane of BBB (Blood Brain Barrier), may change the cerebral vascular permeability and cause the BBB impairment . BBB perUlinastatin is a urinary trypsin inhibitor (UTI) derived from human urine, which can inhibit enzyme activity and effectively reduce systemic inflammatory response . UTI, a This study was a prospective, randomized, double-blind trial approved by the Xuanwu Hospital Ethical Committee (Registration number: ChiCTR-IPR-16008931); written informed consent was obtained from each patient. In this study, we enrolled 60 patients undergoing lumbar discectomy with general anesthesia and 30 age-matched control volunteers to examine the correlation between ulinastatin and serum lipopolysaccharide (LPS), MMP-9, interleukin-6 (IL-6), and C-reactive protein (CRP) levels with early POCD in patients.9/L before surgery, a stroke in the prior 6 months or any other central nervous system diseases, lower digestive diseases, severe deafness or vision problems, illiteracy, and/or communication difficulties related to pronunciation or dialect and postoperative delirium, and refusal or unexpected discharge. We interviewed the patients on the day before surgery and collected baseline data, including age, sex, gender, body-mass index (BMI), past medical history, education history, and MMSE and Montreal Cognitive Assessment (MOCA) score.We enrolled 60 patients older than 65 years (age ≤ 85 years) with ASA status of I-II. Thirty community volunteers older than 65 years (age ≤ 85 years) were examined to exclude the practice effect of repeated neuropsychological testing. Exclusion criteria were: Mini-mental State Examination (MMSE) score ≤23, acute or chronic infectious diseases or trauma, tumor, taking anti-inflammatory drugs or immunosuppressants, WBC ≥ 10000 × 10Before the operation, patients were randomized into a control group (Group C) or a ulinastatin group (Group U). The randomization sequence without stratification was generated by a computer and sealed with consecutively numbered envelopes. The nurses were responsible for preparing ulinastatin or saline placebo with the same 100 ml solution. Patients and investigators were all blinded to group allocation until the final statistical analysis was completed.All patients did not receive sedatives or anticholinergics before anesthesia. Induction of anesthesia was performed in all patients with sufentanil 0.3 μg/kg, etomidate 0.2 mg/kg, and rocuronium 0.8 mg/kg. All patients were intubated and given 50% oxygen by pressure-controlled ventilation. Anesthesia was maintained by an infusion of propofol 3–5 mg/kg/h and remifentanil 0.3 μg/kg/min until the end of operation and sufentanil by intermittent injection (up to 0.5 μg/kg). After the induction of anesthesia, ulinastatin 10,000 units/kg [diluted in normal saline to 100 ml (treatment group)] or 100 ml normal saline (control group) was administered intravenously over a period of 30 min before surgical incision and 5000 units/kg was administered after surgery on the 1st and 2nd days. All patients received intravenous patient-controlled analgesia postoperatively.To measure IL-6, MMP-9, CRP, and lipopolysaccharide (LPS), venous blood samples were obtained at four time points: before the induction of anesthesia (T0), at the end of operation (T1), and 24 and 72 h postoperatively (T2 and T3). Blood samples (4 ml) were centrifuged at 3000 rcf for 15 min at -4°C and stored at -70°C. Cytokine levels of IL-6 and MMP-9 were measured by Enzyme-Linked Immunosorbent Assay . The level of CRP was measured by immunoturbidimetry . The level of LPS was measured by ELISA.Table 1) and obtain the standard deviation (SD), on the 1st day. We compared the scores on the day before operation with the scores on post-operative day 7; we subtracted the practice effect and divided the community volunteers SD (from the 1st day), to obtain a Z score. A patient was considered to exhibit POCD if the Z score was ≥1.96 SD.We used the MMSE test to exclude patients and controls whose score was ≤23; MOCA test was used to examine the cognitive decline level. We compared the MOCA score in community volunteers on the 1st and 8th days. We calculated the average changes to determine the practice effect  between the ulinastatin and control groups were analyzed using Student’s Figure 1. Finally, 60 patients were enrolled and randomized into this study.A total of 103 patients older than 65 years (from 66 to 82 years old) were admitted. Forty-three patients were excluded, which was detailed in Table 2).There were no significant differences between the two groups in characteristics  .There were significant differences in the postoperative decline of the MOCA test. There was a lower incidence of POCD in the ulinastatin group  than in the control group , P < 0.05) .All postoperative data were adjusted to match perioperative data using hematocrit obtained by arterial blood gas analysis. In both control and ulinastatin groups, serum IL-6 concentrations increased at the end of surgery and on post-operative days 1 and 3. However, the ulinastatin group showed lower serum IL-6 concentrations than the control group on post-operative days 1 and 3 (P < 0.05) .In both control and ulinastatin groups, serum CRP concentrations increased on post-operative days 1 and 3. However, the ulinastatin group showed lower serum CRP concentrations than the control group on post-operative day 3 (P < 0.05) .In both control and ulinastatin groups, serum MMP-9 concentrations increased at the end of surgery and on post-operative days 1 and 3. However, the ulinastatin group showed lower serum MMP-9 concentrations than the control group at the end of surgery and on post-operative days 1 and 3 (P < 0.05) .In the control group, serum LPS concentration increased on post-operative day 3. In the ulinastatin group, serum LPS concentration increased at the end of surgery but decreased on post-operative days 1 and 3. The ulinastatin group had lower serum LPS concentration than the control group on post-operative day 3 (P < 0.05) and the adjusted OR  is 0.155 . The crude OR is some bigger than the adjusted OR, but statistical analyses all demonstrated that ulinastatin intervention was a protective factor for POCD (Table 4).The crude OR (without covariates) is 0.262  after the operation compared with the control group; further, there was a lower occurrence of POCD in the ulinastatin group, as assessed by neuropsychological testing. Logistic regression analysis demonstrated that ulinastatin intervention was a protective factor for POCD, which indicated that patients with ulinastatin intervention were not likely to experience POCD. We found that ulinastatin may inhibit the release of serum LPS, IL-6, CRP, and MMP-9, and may prevent the occurrence of POCD. The ulinastatin group had lower serum LPS concentration than the control group on post-operative day 3.in vivo and in vitro . This suggested that ulinastatin may inhibit the release of IL-6. Serum IL-6 is a sensitive marker of tissue damage and is also directly related to the duration of surgery  and the ulinastatin group had lower serum S100β protein and IL-6 concentrations than those in the control group. Our observed effect (ulinastatin group 16% vs. control group 43%) is remarkable. The intervention reduced the incidence of POCD by almost two thirds. Ulinastatin has a broader research perspective and larger-scale studies are urgently needed in the future.Ulinastatin is a protease inhibitor isolated from the urine of healthy adult males. It is not easy to be allergic and safe to use. It can reduce the body’s inflammatory response through various ways such as antioxidant reaction, anti-proteolysis, and inhibition of inflammatory mediator release. Postoperative cognitive disturbance, including postoperative delirium (POD) and POCD, is a common complication of perioperative neurocognition in elderly patients undergoing surgery. POD is characterized by inattention, disorganized thinking, and an altered level of consciousness. Generally, POD mostly occurs in the first 3 days following surgery . POCD isThis study has some limitations. Firstly, the follow-up study time was short. In conclusion, POCD is very common in elderly patients undergoing surgical operations and its mechanism is very complex. In our study, owing to elderly gut fragility, LPS is demonstrated to be a marker for cognitive dysfunction. Ulinastatin can inhibit the release of serum LPS, IL-6, CRP, and MMP-9, and prevent the occurrence of POCD. Further ulinastatin clinical studies about the effect on POCD in people are needed.MZ was the first author of this article, responsible for the design and implementation of this project, data collection, data statistics, and article writing. Y-HZ was responsible for the design and data collection in the project. H-QF was responsible for the article modification. Q-MZ was responsible for the postoperative patient data collection. T-LW was the corresponding author of this article in charge of the project, responsible for the expenses of the project, design, write and check the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CF and handling Editor declared their shared affiliation.


Cell-based therapies with bone marrow-derived progenitor cells (BMDPC) lead to an improved clinical outcome in animal sepsis models. In the present study we evaluated the ability of granulocyte macrophage-colony stimulating factor (GM-CSF) to mobilize BMDPC in a lipopolysaccharide (LPS)-induced sepsis model and thereby its potential as a novel treatment strategy.in vitro.Male Wistar rats received LPS (25μg/kg/h for 4 days) intravenously and were subsequently treated with GM-CSF 12.5μg/kg . As control groups, rats were infused with sodium chloride or GM-CSF only. Clinical and laboratory parameters, proinflammatory plasma cytokines as well as BMDPC counts were analyzed. Cytokine release by isolated peripheral blood mononuclear cells from rat spleen upon incubation with LPS, GM-CSF and a combination of both were investigated In vivo, rats receiving both LPS and GM-CSF, showed a reduced weight loss and increased mobilization of BMDPC. At the same time, this regime resulted in an increased release of proinflammatory cytokines  and a significantly increased mortality. In vitro, the combination of LPS and GM-CSF showed a significantly increased IL-6 release upon incubation compared to incubation with LPS or GM-CSF alone.GM-CSF did not have a beneficial effect on the clinical course in our LPS-induced sepsis model. It synergistically promoted inflammation with LPS and probably thereby impaired survival. Septic shock with multiple organ failure is the leading cause of death in intensive care units and remains a major health problem. Despite extensive research and well-conducted clinical trials, no specific novel treatment strategy exists so far ,2. Since+/AC133+/KDR+), recently also referred to as proangiogenic hematopoietic cells (PHC) , proinflammatory cytokines and flowcytometry.The concentrations of interleukin (IL)-6, IL-8 and vascular endothelial growth factor (VEGF) were assessed in serum using enzyme-linked immunosorbent assay kits  in triplicate samples. Enzyme-linked immunosorbent assays were performed according to the manufacturer’s instructions.+/CD133+-cells have been described and identified as BMDPC in the literature. In brief, one hundred microliters of PBMC (containing 1 x 106 cells) were incubated at 4°C for 30 mins with 10 μL of goat CD34 polyclonal antibodies  and 10 μL of rabbit CD133 polyclonal antibodies . The cells were washed three times to remove unbound antibodies and incubated at 4°C for 30 mins with the secondary antibodies swine anti-rabbit IgG- Fluorescein isothiocyanate isomer 1 (FITC)  and donkey anti-goat IgG (H+L)-Phycoerythrin (PE) . The cells were again washed three times to remove unbound antibodies and finally resuspended in 500 μL of BD FACS lysing solution . Flowcytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using WinMDI 2•8 software . A minimum of 300,000 events were collected. Flowcytometry analysis of each probe was performed in triplicate. The frequency of BMDPC in peripheral blood was determined by a two-dimensional side-scatter/fluorescence dot-plot analysis of the samples after appropriate gating. BMDPC counts are expressed as percentage of total PBMC in each rat.Peripheral blood mononuclear cells (PBMC) were prepared by gradient centrifugation using Ficoll-Hypaque . The expression of cell-surface antigens was determined by two-color immunofluorescence staining using antibodies against CD34 –a marker protein for hematopoietic stem cells- and CD133 –a marker for progenitor cells. CD34The PBMC isolated from rat spleen were prepared by gradient centrifugation using Ficoll-Hypaque . Five specific pathogen free male Wistar rats weighting 450-500g served as organ donors. The rat spleen was first reduced to small pieces and then homogenized using a mesh . The obtained spleen cells were then diluted with phosphate buffer saline 1:4 and pipetted onto the Ficoll solution. Centrifugation was performed at 20°C with 1500 rpm for 30 minutes. After that, the interphase was extracted and purified by 3 successive centrifugation steps . The PBMC were then seeded in 24-well plates in growth medium  and stimulated with GM-CSF  in different concentrations , LPS (0.25μg/ml)  and the combination of both. After 24 hours, IL-6 and cytokine-induced neutrophil chemoattractant-1  were measured in the supernatant using ELISA kits  in triplicate samples. ELISA was performed according to the manufacturer’s instructions.All data are presented as mean ± SEM. The statistical comparisons were carried out by means of one-way ANOVA followed by Holm-Sidak’s post-hoc test as required. The survival rates determined according to Kaplan-Meier were compared with the log rank test. The corrections for multiple comparisons were ensured by the Holm-Sidak test. We considered p<0.05 to be statistically significant. Statistical analyses were performed using SigmaPlot 11.0 .2/FiO2 ratio  was not impaired in any group. On the contrary pH was reduced in the LPS+GMCSF group compared to 0h (p<0.001) and the LPS group (p = 0.006). Ventilation was not compromised, as PaCO2  was not altered. Rather we found a decreased base excess in the LPS (p<0.016) and LPS+GMCSF group p<0.001) compared to 0h. Hemoglobin was reduced in all intervention groups  compared to 0h. There was a also significant difference in hemoglobin between LPS and LPS+GMCSF (p = 0.002).Weight changes were assessed and blood gas analysis was performed at the beginning of the experiment (0h) and at the end (96h). The results are displayed in After 96h we found a statistical significant increase for ASAT (p<0.001), creatinine (p<0.001), urea (p<0.001) and creatine kinase (p<0.001) in the LPS+GMCSF group compared to 0h . In the We used ELISA to detect serum concentrations of IL-6, IL-8 and VEGF in the serum of rats in all the groups after 96 hours . For IL-+/CD133+-BMDPC in the peripheral blood of rats in the sham (n = 2), LPS (n = 3), GM-CSF (n = 3) and LPS+GMCSF group at two time points (0 and 96 h). Our results showed significant differences in the groups  .in vivo-model resulted in increased inflammation markers and mortality, we set out to assess the effect of GM-CSF and LPS alone and in combination on cytokine release in vitro. We isolated PBMC from rat spleen and performed ELISA with the supernatant after 24 hour incubation. The addition of GM-CSF in three ascending dosages to the medium resulted in a moderate increase of IL-6 concentration in the supernatant, although this increase did not reach statistical significance in any of the tested GM-CSF concentrations , LPS (281±24.2pg/ml) and the three different tested concentrations of LPS+GMCSF  compared to the medium (4.34±0.9pg/ml) . However, there has been no statistical difference between the three different concentrations of LPS+ .In vitro, we demonstrated that incubation of isolated PBMC from rat spleen with LPS and GM-CSF simultaneously results in a significantly increased IL-6 release.In previous studies, we and others, have demonstrated that progenitor cells are increasingly mobilized from the bone marrow into the circulation in patients with sepsis  and acutAlready 25 years ago, the use of G-CSF or GM-CSF treatment in animal models of bacteraemia was investigated ,18. Basein vitro by incubating PBMC from rat spleen with LPS and GM-CSF, which has also lead to an increased release of proinflammatory cytokines. It has been shown in the past, that GM-CSF does induce proinflammatory cytokine release, especially IL-6 and IL-8, in macrophages [In contradiction to these results, we have found an increased mortality by GM-CSF application in our sepsis-model. We could demonstrate that animals, which received LPS and GM-CSF, released increasing amounts of pro-inflammatory cytokines and confirmed these results rophages ,20. AlthWith regard to ARDS, there are conflicting results on the effect of GM-CSF. Goodman et al. have demonstrated that plasma from atients with ARDS enhanced PBMC viability, while blocking the GM-CSF receptor significantly reduced PBMC viability in ARDS plasma, but not in normal plasma . They suIn our present study, GM-CSF treatment resulted in an increased mortality of animals. Although BMDPC were increasingly mobilized in response to GM-CSF administration, they were not able to prevent the severe pro-inflammatory effects of the GM-CSF treatment. In line with these observations, levels of G-CSF mRNA correlated with severity of shock, infiltration of polymorphonuclear leukocytes (PMN), pulmonary edema and hypoxia in a rat model of hemorrhagic shock ,25. The Conflicting results in regard to the effect of G-CSF/GM-CSF have also been described for sepsis. In pre-clinical models of sepsis in rats, GM-CSF correlated positively with the survival outcome . A meta-in vivo studies, we also have to discuss the limitations of our experimental set-up. Our endotoxemia model induced by LPS is obviously not identical with an infection caused by a pathogen, in which LPS leads to an activation of the innate immune system to eliminate the pathogen. Also, the survival rate of the animals in the LPS group was hundred percent, which already shows, that the endotoxemia did only cause a mild sepsis. We can only speculate that GM-CSF application could lead to different results in a model of severe sepsis. Another limitation of our set-up relates to the application of GM-CSF simultaneously to the induction of sepsis, which does not reflect the clinical setting, where an application is only possible after sepsis has been manifested. A respective prolonged set-up might have revealed different result. Also, we have not tested a treatment strategy based on G-CSF, which could have also led to a different outcome, since previous studies showed a differing impact by GM-CSF and G-CSF [The limitations of this study are the limited number of experiments and the lack of GM-CSF receptor blockade studies. The results from this study remain therefore associative and loosely mechanistic. To interpret the results of our study within a clinical context and with respect to the design of future nd G-CSF . Anotherin vitro. We have demonstrated that GM-CSF administration not only results in an increased mobilization of BMDPC, but surprisingly impairs survival significantly in our experimental model. There is indication that GM-CSF administration amplifies the LPS-induced release of proinflammatory cytokines, which could also be confirmed


The generic questionnaire WHO-5 Well-being Index (WHO-5), which measures the construct of mental well-being has been widely used in several populations across countries. The questionnaire has demonstrated sufficient psychometric properties; however, the test- retest reliability of the WHO-5 scale has yet to be determined. The aim of this study was to evaluate the test-retest reliability and measurement error of the Danish WHO-5 Well-being Index for outpatients with epilepsy. A further aim was to evaluate whether the method of administration  influenced the results.Epilepsy outpatients aged ≥15 years from three outpatient clinics in Central Denmark Region were included from August 2016 to April 2017. The participants were randomly divided into four test-retest groups: web-web, paper-paper, web-paper, and paper-web. Test-retest reliability was assessed by intraclass correlation coefficients (ICC) and measurement error by calculating minimal detectable change (MDC) on the basis of the standard error of the measurement.A total of 554 patients completed the questionnaire at two time points. The median duration between test-retest was 22 days. The pooled test-retest reliability estimate was ICC 0.81 . The estimated MDC was 23.60 points . These estimates showed little variation across administration methods.WHO-5 showed acceptable test-retest reliability in a Danish epilepsy outpatient population across different method of administration; however, the relatively large measurement error should be taken into account when evaluating changes in WHO-5 scores over time. Further research should be done to explore these findings. Several considerations are important when selecting patient-reported outcome (PRO) measures for use in clinical practice. A PRO measure should be relevant to patients and clinicians and possess an adequate level of psychometric evidence for the instrument in the target population . In CentWHO-5 is a generic unidimensional questionnaire reflecting the construct mental well-being during the last 2 weeks . WHO-5 iThe study aim was to evaluate the test-retest reliability and measurement error of the Danish WHO-5 Well-being Index for outpatients with epilepsy. A further aim was to evaluate whether the method of administration  influenced the results.Patients with epilepsy aged ≥15 years from three outpatient clinics in Central Denmark Region were included from August 2016 to April 2017. The patients completed the questionnaire at two time points. First, they completed a questionnaire from the outpatient clinic based on their preferred web or paper administration method (test 1). Subsequently, approximately 2 weeks later, a letter was sent to the patients asking them to complete the same questionnaire again (test 2). The patients were randomly divided into four test-retest groups based on the method of administration at test 1 and test 2: web-web, paper-paper, web-paper, and paper-web. Three reminders were sent in test 1, but no reminders were sent to non-responders in test 2. The WHO-5 Well-being Index was included in the questionnaire in test 1. In addition, the questionnaire included other items, regarding, for example, seizures, symptoms, and general health. The general health construct was measured by using two items from the Danish version of the Short Form 36 Health Survey . A long 95 equals 1.96 ± √2 x SEM and indicates the smallest within-person change that can be interpreted as a “real” individual change above the measurement error [95can be attributed to measurement error [Descriptive statistics were generated for patient characteristics and for each item to determine the extent of missing values and floor- or ceiling effects, which were considered present if more than 15% had a score at the lower or upper end of the scale . Cronbacnt error . Thus, ant error . PatientA total of 554/1640 (34%) patients responded to the questionnaire twice. The median age was 57.3 years Table . Cronbac95 of 23.60 points . The analysis was repeated in the four test-retest groups . This may be due to the pragmatic design, which allowed patients to choose administration method for their response to test 1. In the Danish general population, a mean WHO-5 score of 70 points has been reported , 27. ThiThe WHO-5 Well-being Index showed acceptable test-retest reliability in a Danish epilepsy outpatient population, but the measurement error of the scale was relatively large. Different methods of administration did not influence the results. Further studies are required to provide insight into the test-retest reliability and measurement error in different language versions of the WHO-5 Well-being Index and in different patient populations.


The primary endpoint was comparison of the total naso-ocular symptom score (TSS). Secondary endpoints including the increment cost effective ratio (ICER) were also determined. Continuous pollen dispersion began on the 24th of February. Therefore, Group A and Group B received 3-weeks and 1-week of prophylactic treatment, respectively, whereas Group C received post-onset treatment. During the peak pollen-dispersal period, significant differences in TSS were seen between the groups, particularly between Group A and C. The ICER of Group B vs. Group C was lower than that of Group A vs. Group C. These results suggest that long-term prophylactic treatment with FFNS is clinically the most potent treatment, whereas short-term prophylactic treatment is cost effective for pollen-induced allergic rhinitis.Prophylactic treatment with intranasal corticosteroids is effective for pollen-induced seasonal allergic rhinitis. However, the appropriate time to start this treatment remains unclear. We performed a double-blinded, randomized, placebo-controlled trial. Starting on 1 February 2014, patients with Japanese cedar pollinosis received either fluticasone furoate nasal spray (FFNS) for 8 weeks (Group A:  Prophylactic treatment, an early interventional or initial treatment, is recommended for patients with pollinosis who annually experience substantial naso-ocular allergic symptoms during the pollen season . This trPrevious studies have reported starting prophylactic treatment with INS at time points ranging from 4 weeks before the pollen season to the beginning of pollinosis symptoms ,9,10,11.In order to clarify the appropriate time to start treatment, we performed a three-armed, double-blinded, randomized, placebo-controlled trial using a fluticasone furoate nasal spray (FFNS) for JCP. These results, including economic evaluation, provide a basis for the use of INS for prophylactic treatment in pollen-induced AR.Inclusion and exclusion criteria for the JCP subjects enrolled in this study were identical to those in our previous reports ,11. In bThe study was a single-center, double-blinded, randomized, placebo-controlled, three-armed parallel group trial that was performed from 2013 to 2014. The primary endpoint was comparison of the total naso-ocular symptom score  that were obtained from diary cards filled out from January 27 to 28 March in 2014 . The secScreening visits were performed in December 2013. Prior to the study initiation, we estimated the sample size that would be required based on the mean and standard deviation values in the groups reported in our previous studies using INS ,11. In on = 24), a placebo nasal spray for 2 weeks followed by FFNS for 6 weeks (Group B: n = 23), or placebo for 4 weeks followed by FFNS for 4 weeks (Group C: n = 23). The placebo spray that was provided by GSK  had a white-colored lid and trigger. The lid and trigger of the FFNS bottle, which were initially cyan-colored, were changed to white to ensure that both FFNP and the placebo spray had the same appearance. Patients were allowed to use a levocetirizine tablet and olopatadine eye drops as rescue medications on demand. The study was approved by the Institutional Review Board of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences . Written informed consent was obtained from each patient.Allocation concealment was granted by the central registry and computer-generated block randomization. Starting on 1 February 2014, patients received either FFNS (55 μg per nostril once a day in the morning) for 8 weeks  to post-onset treatment (Group C) was compared between the long-term (Group A) and the short-term (Group B) prophylactic treatments . Effecti2 was counted [From January 7, 2014, daily Japanese cedar and Japanese cypress pollen counts were performed using Durham samplers that were installed on the rooftops of the Okayama University Hospital buildings. The first day of the major pollen period was defined as the first of two consecutive days for which >1 grain/cm counted .p < 0.0.5 considered to be significant.Values are expressed as the median for each subject group. A non-parametric Kruskal-Wallis test followed by a Dunn’s test was used to compare the data between groups. Statistical analyses were performed using GraphPad Prism  version 9.2, with Out of the 72 patients that were screened, a total of 70 patients fulfilled the inclusion criteria. After enrollment in the study, patients were randomized into one of three groups, with 24 (Group A) or 23 (Group B and C) patients per group. Group A received FFNS for 8 weeks; Group B received placebo for 2 weeks flowed by FFNS for 6 weeks; and Group C received placebo for the first 4 weeks followed by FFNS for 4 weeks. Sixty-nine subjects (98.6%) completed the study. One patient in Group A dropped out due to de novo pregnancy. Therefore, the full analysis set consisted of 69 patients (23 patients in each group). The treatment groups were comparable with respect to demographic characteristics, which included age, sex, disease duration, and severity of Japanese cedar/cypress pollinosis (JCCP) .2/day. The total cedar pollen count during the study period was 1419.3 grains/cm2  of the Japanese cedar pollen occurred on January 17, with major continuous dispersion beginning on 24 February. The highest cedar dispersion was seen on 17 March, with 374.3 grains/cmains/cm2 . The trep < 0.05 by a Kruskal-Wallis test). In addition, a Dunn’s test indicated that Group A showed significant symptom alleviation on 3 days (13–15 March) as compared with Group C (p < 0.05). A clinically important difference (more than 1.5 points) was also seen between Group A and Group C . Although a Dunn’s test showed no significant differences between the groups, a clinically important difference (more than 0.9 point) between Group A and B and between Group A and C was seen on the latter day in the peak pollen dispersal period    A.p < 0.05). A Dunn’s test further indicated a significant symptom alleviation in the prophylactic treatment groups (Group A and Group B) as compared with the post-onset treatment group (Group C) (both p < 0.05)  also showed a significant difference between the three groups ( < 0.05) B. In qua < 0.05) .p < 0.05). A Dunn’s test further indicated a significant fewer drug consumption in the prophylactic treatment groups (Group A and Group B) as compared with the post-onset treatment group (Group C) (both p < 0.05) (p < 0.05 by a Dunn’s test)  showed a significant difference on 14 March between the groups ( < 0.05) C. A simi’s test) D.Group A took 2.8 levocetirizine dihydrochloride tablets (315 yen/tablet) and 1.00 olopatadine hydrochloride eye drops bottle (198 yen/bottle), Group B took 2.4 levocetirizine dihydrochloride tablets and 0.96 olopatadine hydrochloride eye drops bottle, and Group C took 4.5 levocetirizine dihydrochloride tablets and 1.43 olopatadine hydrochloride eye drops bottle . No sympp < 0.05)  A,D,E. No < 0.05) B,C. DuriAll the treatments were well-tolerated and had a similar safety profile . While tThe sum score of the TNSS over the study period for Group A, B and C was 130, 150 and 191, respectively. The medication cost for Group A, B and C was 8673, 6633 and 5001 yen, respectively . The ICEThe present study demonstrated a significant difference in the control of pollinosis between the prophylactic and the post-onset treatments with FFNS. In particular, the 3-weeks of prophylactic treatment with FFNS showed a substantial effect on the control of nasal symptoms as compared with the post-onset treatment. On the other hand, the present study clarified for the first time that the ICER for the post-onset treatment was lower for the 1-week prophylactic treatment with FFNS than that for the 3-weeks of treatment. These results suggest that long-term prophylactic treatment with FFNS is clinically the most potent treatment, especially for nasal symptoms, whereas short-term prophylactic treatment is cost effective for Japanese cedar pollinosis.Although prophylactic treatment with INS is effective and safe for pollinosis, little is known about the appropriate time to start this treatment ,9,10,11.It is suggested that INS has good effects on sneezing, and INS was effective for ocular itching through the naso-ocular reflex. Also, during the preseanoal priod (around 8 February), there was a significant difference in symptom scores among the group. It is suggested that in the beginning of February a small amount of pollen dispersion was found and the patients might have been affected by pollen stimulation.One of the bases of prophylactic treatment with INS for pollinosis is the control of pre-onset minimal persistent inflammation that is present in the initial dispersion of pollen prior to the onset of symptoms, and which contributes to hyperreactivity and subsequently to the onset of full-scale symptoms ,5. We haIntranasal corticosteroids reduce ocular symptoms such as watery and itchy eyes through several mechanisms including reduction of nasal-ocular reflexes ,15. We hEconomic evaluation of new medical interventions is of growing concern due to limited health care resources. Economic evaluation includes cost minimization analysis, cost-effective analysis, cost-utility analysis and cost-benefit analysis . Of thesSince the present study was designed as a three-armed parallel group trial, the ICER of 1-week and 3-week prophylactic treatment with FFNP could be compared to post-onset treatment. Our results showed that the ICER of the 1-week prophylactic treatment to post-onset treatment was lower than that of the 3-week prophylactic treatment, which suggested that short-term prophylactic treatment with INS is a cost-effective therapy for controlling allergic symptoms in JCP. Without placebo treatment, the symptom score might have been worse, and therefore, cost effectiveness difference might have been more significant in the 3-week prophylactic treatment group compared to the post-onset treatment group. On the other hand, our study has a limitation in terms of cost estimation because the cost did not include indirect medical costs such as loss of productivity or intangible costs such as mental anguish . Direct Long-term prophylactic treatment with FFNS is clinically the most potent treatment, whereas short-term prophylactic treatment is more cost effective for Japanese cedar pollinosis. These results may provide a basis for determination of the appropriate time to start prophylactic treatment in pollinosis.


Cognitive dysfunction is one of the serious complications induced by status epilepticus (SE), which has a significant negative impact on patients’ quality of life. Previous studies demonstrated that the pathophysiological changes after SE such as oxidative stress, inflammatory reaction contribute to neuronal damage. A recent study indicated that preventive astaxanthin (AST) alleviated epilepsy-induced oxidative stress and neuronal apoptosis in the brain. In the present study, rats were treated with vehicle or AST 1 h after SE onset and were injected once every other day for 2 weeks . The results showed that the cognitive function in SE rats was significantly impaired, and AST treatment improved cognitive function in the Morris water maze (MWM). Magnetic resonance imaging (MRI), hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining showed obvious damage in the hippocampus of SE rats, and AST alleviated the damage. Subsequently, we evaluated the effect of AST on relative pathophysiology to elucidate the possible mechanisms. To evaluate the oxidative stress, the expression of malondialdehyde (MDA) and superoxide dismutase (SOD) in plasma were detected using commercially available kits. NADPH oxidase-4 (Nox-4), p22phox, NF-E2-related factor 2 (Nrf-2), heme oxygenase 1 (Ho-1) and sod1 in the parahippocampal cortex and hippocampus were detected using western blot and real-time polymerase chain reaction (RT-PCR). The levels of MDA in plasma and Nox-4 and p22phox in the brain increased in SE rats, and the levels of SOD in plasma and Nrf-2, Ho-1 and sod1 in the brain decreased. Treatment with AST alleviated these changes. We also detected the levels of inflammatory mediators like cyclooxygenase-2 (cox-2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and NF-κB phosphorylation p65 (p-p65)/p65 in the brain. The inflammatory reaction was significantly activated in the brain of SE rats, and AST alleviated neuroinflammation. We detected the levels of p-Akt, Akt, B-cell lymphoma-2 (Bcl-2), Bax, cleaved caspase-3, and caspase-3 in the parahippocampal cortex and hippocampus using western blot. The levels of p-Akt/Akt and Bcl-2 decreased in SE rats, Bax and cleaved caspase-3/caspase-3 increased, while AST alleviated these changes. The present study indicated that AST exerted an reobvious neuroprotective effect in pilocarpine-induced SE rats. Status epilepticus (SE) is one of the most common neurological emergency diseases, which is ascribed to the failure of the mechanisms responsible for seizure termination or the initiation of mechanisms leading to abnormally prolonged seizures. Accumulating evidence demonstrated that SE results in extensive brain damage or death  and some anesthetics, which control approximately two-thirds of seizures. However, these drugs often exacerbate comorbidities, such as cognitive impairment /anti-oxidant response element (ARE) antioxidant pathway  is a lutein carotenoid, which is widely found in a variety of micro- and marine organisms, such as algae, yeast, salmon, trout, krill, shrimp and crayfish. Previous studies demonstrated that AST was a powerful antioxidant because of its peculiar molecular structure of a conjugated double bond at the center and unsaturated ketonic groups at both ends of the aromatic nucleus  were used in the study and were purchased from the Animal Center of Fudan University . All rats were raised in a standard animal-grade room with four or five rats in each cage. This study was carried out in accordance with the recommendations of Guide for Animal Experiments of the Chinese Academy of Medical Sciences. The protocol was approved by the ethics committee for animal care of Jinshan Hospital of Fudan University. The housing environment met the following conditions: ambient temperature approximately 23 ± 2°C, relative humidity (60 ± 5%) and a 12 h light/dark cycle with free access to food and water. All animal experiments were performed during the light cycle. All rats were housed in the laboratory for 1 week prior to experimentation.Male Wistar rats  and pilocarpine  were used to induce SE . AST  was dissolved in vehicle mixing 1:1 polyethylene glycol  and tri-distilled water. The AST group was intraperitoneally injected with 30 mg/kg AST , according to previous studies  = −2.2 mm, lateral (L) = ±3.2 mm] under anesthesia with 10% chloral hydrate . Two other stainless steel screws were implanted in cerebellar regions as a grounding electrode and reference electrode. The screws were fixed with dental cement . Modeling began 7 days after electrode implantation.MRI was performed under anesthesia with 10% chloral hydrate . We collected high-resolution T2-weighted phase (T2WI) images of the hippocampus. RARE sequence was used to detect the MRI of the hippocampus of rats using the following parameters: repeat time (TR): 6,000 ms; echo time (TE): 13.7 ms; matrix size: 152 × 152; magnetic resonance FOV: 26 × 26 mm; MRI scanning layer thickness: 0.5 mm; and acquisition time: 3 min 15 s.The MWM consisted of a circular pool in which rats were trained to escape from the water by swimming to a hidden platform, as described previously , washed, dehydrated, transparentized, immersed in wax, and cut into slices. The dried sections were immersed in a dyeing vessel containing xylene I and xylene II for dewaxing. Sections were successively immersed in different concentrations of alcohol, double-distilled water to hydrate, stained in HE, dehydrated, and sealed. The prepared tissue sections were observed under an optical microscope and photographed using a microimaging system.The brain tissues of rats were fixed with 4% paraformaldehyde , washed, dehydrated, transparentized, immersed in wax, and cut into slices. The dried sections were immersed in a dyeing vessel containing xylene I and xylene II for dewaxing. The sections were hydrated, digested with trypsin, and stained in TUNEL mix. The sections were dehydrated, transparentized and sealed with neutral gum. The CA1 region of the hippocampus was observed and analyzed using an optical microscope to calculate the apoptosis rate.Assays of the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity involved commercially available kits . Plasma was diluted with saline to an appropriate concentration to estimate the MDA content and SOD activity. All procedures were performed under the guidance of the manufacturer’s instructions. MDA levels were measured using a 2-thiobarbital acid method. Optical density was measured at 532 nm. The results were expressed as nmol/mg protein. SOD activity was determined based on its ability to inhibit the oxidation of superoxide anions produced by a xanthine-xanthine oxidase system. One unit of SOD activity was defined as a 50% reduction of optical density at 450 nm absorbance. The results were expressed as U/ml. Colorimetry was used for determination, and the absorbance values and standard equation were used for the assay.Hippocampal and cortical tissue proteins were extracted using a lysis buffer containing 1% PMSF . Protein content was determined using the BCA assay . Proteins (20 μg) were separated using SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA/TBST or skim milk at room temperature for 1 h. The membranes were washed with TBST and incubated with a relatively specific primary antibody  at 4°C oTM reagent kit . Primer Express software v3.0.1 was used to design the RT-PCR primers. SYBR Premix Ex Taq  was used to perform RT-PCR in the Applied Biosystem 7300 , to detect the levels of β-actin and target genes. The 2−ΔΔCt method was used to assess the levels of relative mRNA normalized to β-actin. The primer sequences were shown in The total RNA of hippocampal and cortical brain tissue was extracted using the Trizol Reagent kit . All procedures were performed under the guidance of the manufacturer’s instructions. cDNA of the mRNA of target genes was synthesized using the PrimeScriptt-test was used to compare the differences between the two groups. The escape latency of the MWM was analyzed using repeated measures ANOVA, with factors of strain and days. P < 0.05 was used to indicate significant differences. All experiments were repeated at least three times.The results were expressed as the mean value ± SD. All data analyses were performed using GraphPad Prism 7. The paired p < 0.01 vs. Normal). However, the escape latency in the AST group was reduced compared to the SE group . In contrast, AST reduced the apoptosis rate .TUNEL staining showed that AST intervention ameliorated SE-induced apoptosis of hippocampal neurons, and AST intervention ameliorated the apoptosis of neurons in the CA1 region of the hippocampus. As shown in via the Nrf-2/ARE pathway. p < 0.05), and the SOD level decreased (p < 0.05). AST downregulated MDA (p < 0.05) level and upregulated SOD level (p < 0.05) in the plasma of SE rats. These results suggested that AST reduces the systemic oxidative stress response and enhances antioxidant capacity in SE rats. To evaluate the level of oxidative stress in the brain, we assayed the expression of oxidant indicators including p22phox and NADPH oxidase-4 (Nox-4) in the hippocampus and parahippocampal cortex of rats. Nox-4 is an NADPH oxidase, and p22phox is a subunit of NADPH oxidase. Both enzymes are major sources of reactive oxygen species (ROS). We detected the levels of protein and mRNA using western blot and RT-PCR, respectively. As shown in p < 0.05 vs. Normal; **p < 0.01 vs. Normal; #p < 0.05 vs. SE; ##p < 0.01 vs. SE). The levels of Nrf-2, heme oxygenase 1 (Ho-1) and sod1, which are major molecules of the antioxidant Nrf-2/ARE pathway, were detected using western blot and RT-PCR. The results showed that the levels of Nrf-2, Ho-1 and sod1 protein and mRNA in the hippocampus and parahippocampal cortex of rats in the SE group significantly decreased compared to the Normal group , and AST significantly elevated the expression of Nrf-2, Ho-1 and sod1 , interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), in the hippocampus and parahippocampal cortex of rats using western blot and RT-PCR. As shown in p < 0.05 vs. Normal; **p < 0.01 vs. Normal). However, AST intervention significantly reduced the expression levels of these inflammatory factors compared to the SE group . To investigate the effects of AST on the NF-κB pathway, we also detected the levels of NF-κB p65 and phosphorylation p65 (p-p65) using western blot. The results showed that the expression of p-p65/p65 in the brain of rats in the SE group significantly increased compared to the Normal group , and AST reduced the expression of p-p65/p65 , which suggests the activation of apoptosis. AST reduced the expression of these proteins (p < 0.01). We detected the levels of molecules associated with the PI3K/Akt pathway using western blotting. B-cell lymphoma-2 (Bcl-2) associated X protein (bax) levels in the SE group increased and the levels of phospho-Akt/Akt and Bcl-2 decreased compared to the Normal group . AST reduced Bax expression and increased the expression of p-Akt/Akt and Bcl-2 (##p < 0.01 vs. SE; The results suggested that AST alleviated SE-induced neuronal apoptosis poptosis . We deteEarly pathophysiological insults of SE in rodents include imbalanced oxidative stress, intense neuroinflammatory responses, selective neuronal degeneration, and disruption of the brain-blood barrier, which result in subsequent cognitive impairment Walker, . The lonCognitive dysfunction is a common clinical manifestation of SE-induced brain injury. Long-term epileptic seizures inevitably affect the cognitive function of patients with SE and compromise the quality of life of these patients (Power et al., via reducing oxidative stress and enhancing anti-oxidant capacity.Numerous studies demonstrated that inhibition of oxidative stress induced by seizure was enormously valuable. Activated oxidative stress in epilepsy and other neurological diseases accelerated chronic neuronal dysfunction and neurodegeneration. Oxidative stress activated by epileptic seizure may be a key incentive for recurrent seizures and exacerbate the consequences of seizures, such as neuronal death and cognitive dysfunction, and oxidative stress itself also results in neuronal death (Pearson-Smith and Patel, in vitro experiments and animal models that inhibition of the inflammatory response provides a significant neuroprotective effect and reduces the seizure degree and mortality (Serrano et al., via multiple pathways (Kim et al., via the NF-κB pathway.Burgeoning evidence from recent research studies considerably highlighted the key role of the inflammatory response in the pathogenesis of epilepsy. Inflammatory responses promote the occurrence and development of epilepsy. The brain tissue of patients with epilepsy undergoing surgical resection exhibits a strong inflammatory response, including reactive astrocyte proliferation, the infiltration of activated microglia, and the upregulation of various pro-inflammatory factors, including Cox-2, IL- 1β, TNF-α, and IL-6. Inflammatory cells, including mononuclear cells and neutrophil granulocytes, also increase in the brain, and inflammatory cells infiltrate from the periphery, which can promote inflammatory reactions in the brain and aggravate local neuronal injury (Varvel et al., via regulating the expression of apoptosis-related molecules (Jin et al., via the PI3K/Akt pathway.SE resulting in neurodegeneration, especially hippocampal neurons, is a prominent feature of clinical epilepsy Dam, . SE contIn brief, the present study demonstrated that AST exerted significant neuroprotective effects on neurological damage after SE. The data highlighted that AST afforded neuroprotection after a delayed post-injury intervention. AST blocked the rapid onset oxidative stress, inflammatory pathway and neuronal death during SE. AST, or co-treatment with AEDs, is likely to be a promising strategy to improve treatment efficacy.All datasets generated for this study are included in the manuscript.XD performed the western blot and RT-PCR, generated the figures, and wrote the manuscript. SH and YS performed MRI, H&E staining and TUNEL staining of hippocampus. YF, YX and MWu established the SE models and performed EEG. MWa and XD performed the Morris water maze experiments. YC and XS contributed to the design and performance of experiments and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Serum hemeoxygenase-1 (HO-1) has been proposed to be a biomarker of lung disease activity and prognosis. The present study aimed at evaluating whether HO-1 could be a useful marker for evaluating disease activity and predicting prognosis in patients with interstitial pneumonia (IP).  Serum HO-1 levels of newly diagnosed or untreated patients with IP were measured at hospitalization. We evaluated the relationships between serum HO-1 and other serum biomarkers, high resolution CT (HRCT) findings, and hospital mortality. p < 0.001), and the best cut-off level to discriminate between having an AE or not having an AE was 41.6 ng/mL. Serum HO-1 levels were positively correlated with serum levels of surfactant protein-D  and the ground glass opacity score . Patients who subsequently died in hospital had presented with significantly higher HO-1 levels than those who did not die in hospital .  Twenty-eight patients with IP, including 14 having an acute exacerbation (AE) and 14 not having an AE, were evaluated. The patients having an AE had significantly higher HO-1 levels than those not having an AE (53.5 ng/mL vs. 24.1 ng/mL;  Serum HO-1 may serve as a useful biomarker for detecting AE or predicting hospital mortality in patients with IP. Hemeoxygenase-1 (HO-1) is a 32 kDa heat shock protein that converts heme to carbon monoxide, iron, and bilirubin –7. AdditThis study enrolled a total of 28 newly diagnosed and untreated IP patients who, from April 2011 to March 2018, had been admitted to the hospital and were able to provide informed consent for participation in this research. The extracted data included the patients' medical histories, the physical examination findings, the results of blood biomarkers including an arterial blood gas analysis, and the high-resolution CT (HRCT) findings. The diagnosis of idiopathic pulmonary fibrosis (IPF) was based on the IPF consensus classification . In the KL-6 , and partial pressure of oxygen in arterial blood (PaO2)/a fraction of the inspiratory oxygen (FiO2) (PaO2/FiO2 ratio). Serum HO-1 levels were measured using the ImmunoSet™ HO-1 ELISA development set , according to the manufacturers' instructions. The details of this ELISA method have been described previously [Blood samples were obtained at hospitalization from each patient. We measured serum HO-1 along with lactate dehydrogenase , surfactant protein- (SP-) A , SP-D normal <110 ng/mL), 0 ng/mL, The HRCT findings were evaluated using the semiquantitative scoring method described by Ooi et al. . The lunth to 75th percentiles unless otherwise indicated. Statistical analysis was performed using JMP11 . Group comparisons were made using Wilcoxon's rank-sum test or the chi-squared test. We performed a receiver operating characteristics (ROC) curve analysis to determine the most suitable cut-off levels of serum HO-1 and other blood biomarkers for detecting an AE and predicting hospital death among patients having an AE. Nonparametric Spearman's rank correlation coefficients were calculated to assess the correlations between the serum HO-1 levels and other clinical parameters. p < 0.05 was considered significant.All data are expressed as the median with 25All participants provided informed consent prior to participation in this research. All aspects of the study were approved by the Institutional Review Board of Yokohama City University Medical Center , National Defense Medical College , and Yokohama City University Graduate School of Medicine .2/FiO2 ratios, and GGO scores calculated from HRCT than those not having an AE.Clinical characteristics of patients with IP are summarized in p < 0.001) and in control subjects  (p < 0.001) and in control subjects   . Among p< 0.001) .The ROC curve analysis for the serum HO-1 level was evaluated to discriminate the patients having an AE from the patients not having an AE. The area under the ROC curve (AUC) was 0.91, and the best cut-off level was 41.6 ng/mL. Using this cut-off level, serum HO-1 had a sensitivity of 92% and a specificity of 82% for detecting an AE. An AUC for HO-1 was higher than that obtained for other blood biomarkers and HRCT scores .r=0.66, p < 0.001) and between the serum HO-1 and the GGO score   and between the serum HO-1 and the GGO score   (p=0.053)  days. Patients who subsequently died in hospital had presented with significantly higher HO-1 levels than those who did not die in hospital  . Among pp=0.053) . The ROCp=0.053) .Our preliminary histological data showed that alveolar macrophages in the AE of an IPF patient (autopsy case) appeared to possess higher HO-1 expression than did macrophages in a stable IPF patient  .Serum HO-1 has been proposed as a marker for lung disease prognosis. HO-1 generates biliverdin IXα, ferrous iron, and CO from the oxidation of heme, and exhaled CO reflects active heme metabolism . We prevSamples from fibrotic ILD patients having an AE exhibit diffuse alveolar damage superimposed on chronic fibrosis when evaluated by histology –16. We fIn the presence of alveolitis, the surfactant apoproteins  are secreted by type II pneumocytes; these apoproteins can be detected in the serum as a biomarker of alveolitis , 21. Ser2/FiO2 ratio , and extent of abnormal HRCT findings. Using a single serum biomarker, it may be difficult to estimate the disease prognosis of a patient having an AE; however, we found that the patients who subsequently died in hospital had presented with significantly higher HO-1 levels than those who did not die in hospital. This result is consistent with our previous observation, in patients with ARDS, that serum HO-1 levels during intensive care unit stays were persistently higher in individuals who subsequently died (compared to those of survived these incidents) [Biomarkers for estimating the disease prognosis of the patients having an AE are not well established in the clinical setting. A previous study reported a composite scoring system for patients with AEs . This sccidents) . TherefoWe speculate that serum HO-1 may serve as a useful biomarker for detecting an AE, for evaluating lung inflammation, and for predicting hospital mortality among patients with IP. However, the research described here does include several limitations. First, ours was a single-center study with a small number of patients. This work will need to be expanded to a multicenter prospective study to evaluate the reproducibility of these results. Second, the clinical diagnoses of enrolled patients were heterogeneous, although there was no significant difference between the AE group and the non-AE group. Future research will need to evaluate the clinical utility of serum HO-1 measurement in patients with each of the various histopathological diagnoses .


A growing body of research has demonstrated that the degeneration of chondrocytes is the primary cause of osteoarthritis (OA). Parathyroid hormone-related protein (PTHrP) can alleviate the degeneration of chondrocytes via promotion of chondrocyte proliferation and inhibition of terminal differentiation, but the underlying mechanism remains unknown. This study aimed to identify the microRNAs (miRNAs) that may target PTHrP and regulate the proliferation and terminal differentiation of chondrocytes. Bioinformatic analysis was used to predict which miRNAs target PTHrP. We collected human knee cartilage specimens to acquire the primary chondrocytes, which we then used to test the expression and function of the targeted miRNAs. To explore the effects of miR-15a-5p on the putative binding sites, specific mimics or inhibitors were transfected into the chondrocytes. Furthermore, a dual-luciferase reporter gene assay and chondrocyte degeneration-related factors were used to verify the possible mechanism. The expression of PTHrP was upregulated in the OA chondrocytes, whilst miR-15a-5p was downregulated in the OA chondrocytes. A negative correlation was observed between PTHrP and miR-15a-5p. The knockdown of miR-15a-5p promoted the growth of chondrocytes and inhibited calcium deposition, whilst overexpression of miR-15a-5p reversed this trend. The effect of miR-15a-5p overexpression was neutralised by PTHrP. Dual-luciferase reporter assays revealed that PTHrP can be used as a novel targeting molecule for miR-15a-5p. miR-15a-5p promotes the degeneration of chondrocytes by targeting PTHrP and, in addition to helping us understand the development of OA, may be a potential biomarker of OA. Osteoarthritis (OA), a degenerative joint condition, is the most common disease in adults and, in addition to leading to high medical expenses, is one of the most frequent causes of pain, loss of function, and psychological disability , 2. SeveDeterioration of the smoothness of the articular cartilage is the chief characteristic of OA, and chondrocytes are the only type of cells in the articular cartilage, so dysregulation of cell proliferation and differentiation in chondrocytes contributes directly to OA –7. The pParathyroid hormone-related protein  is a widely occurring protein produced by most tissues in the body , 9 and iMicroRNAs (miRNAs), about 23 nt of endogenous small noncoding RNA, are involved in the regulation of target protein-encoding genes via translational inhibition and/or degradation of the target mRNA –16. The In this study, rheumatoid arthritis and septic arthritis were excluded, and samples of human articular cartilage were obtained from 16 patients with OA (OA Grade III–IV) who underwent total knee arthroplasty. Eight patients with osteosarcoma who were undergoing segmental resection and artificial knee prosthesis replacement or amputation were also recruited to allow collection of normal articular cartilage specimens. 3 pieces with two sterile scalpels. The PBS was removed, and the diced tissues were placed in Dulbecco's modified Eagle's medium  with 2.0 mg/mL collagenase type II . The tube was capped, covered with Parafilm, and then placed on an orbital shaker at 200 rpm for 10–12 h at 37°C. The next day, the suspension was filtered through a 40-µm mesh into a sterile 50-ml centrifuge tube. The digestate was centrifuged at 1200 rpm for 10 min. The supernatant was removed, and the pellet was gently resuspended in 10 mL of DMEM. The specific protocol of the primary chondrocyte culture is based on the literature [2. The chondrocytes in the second passage were used in all of the experiments.The cartilage specimens were collected immediately after surgery, placed in Petri dishes, and wetted with phosphate-buffered saline solution (PBS). The cartilage samples were cut into 1–3 mmterature , 22. Theµm. Serial sections from each slice were stained routinely with Safranin O/Fast Green. The cell-attached slides were washed using PBS and stained using Toluidine blue for 15 min. The cells were rewashed with PBS, and the morphology of the cartilage tissues and cells was assessed.The excised cartilage tissues were fixed in 4% paraformaldehyde (PFA), briefly decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 4 weeks, and sectioned into 4–5 mm thick slices with a scalpel blade. Each slice was embedded in paraffin and sectioned at 6 After being seeded into special chamber slides, the human primary chondrocytes were fixed with 4% PFA for 10 min, permeabilised with 0.1% Triton X-100 for 15 min, and treated with 1% bovine serum albumin for 30 min. Next, the cells were incubated with PTHrP antibody for 1 h, and the anti-goat IgG antibody conjugated with peroxidase was then incubated for 30 min. Finally, the nuclei were counterstained with haematoxylin. All of the procedures were performed at room temperature.To evaluate the extent of the calcification or calcium deposition in the intervention and control groups of the cultured primary chondrocytes, we used Alizarin Red S staining, which is one of the most commonly used methods. The cells were fixed at room temperature with 4% PFA for 10 min and exposed to Alizarin Red S for 15 min. The cells were then washed with PBS and then photographed under a microscope.The total protein was extracted from each intervention group by lysing the cells in a radioimmunoprecipitation assay buffer . Quantitative analysis of the protein concentrations was performed with a special analytical kit . The same amount of proteins was obtained by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked with 5% skimmed milk powder in TBST  and cultured with primary and secondary antibodies. The specific protocol of the western blotting is based on the literature . After wOA chondrocytes in logarithmic phase were seeded into six-well plates and cultured for 24 h. The cells were then allotted to several groups: the siPTHrP group, the negative control (NC) group, the miR-15a-5p mimics group, the NC inhibitors group, and the miR-15a-5p inhibitors group. The plasmids were purchased from GenePharma Co Ltd . The cells were transiently transfected with plasmids using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) as previously reported [We constructed wild-type and mutant plasmids for PTHrP to explore the targeting link between miR-15a-5p and PTHrP. HEK293T cells were added into a 12-well plate to confirm the direct interaction between miR-15a-5p and PTHrP mRNA. The cells were transfected with miR-15a-5p mimics and PTHrP wild-type or PTHrP mutants using the Lipofectamine 2000 reagent after 24 hours. The samples were then cotransfected with a pRL-TK plasmid that expresses Renilla luciferase. About 48 hours after the transfection, the cells were lysed and the Firefly and Renilla luciferase activities were detected with a dual luciferase reporter assay system . The transfection was performed in triplicate.The total RNA was separated by Trizol Reagent (Thermo Fisher Science), reverse transcribed into cDNA, and used for the qRT-PCR analysis, as previously reported . The ABIt-test was used to detect the differences between the two groups. A P value of less than 0.05 was considered to indicate significant variation between groups.The statistical analysis was performed using GraphPad Prism Software (Version 7.0). The results are expressed as mean ± standard deviation (SD). Student's p<0.01) ( (p<0.05)  . Analysi(p<0.05) .p<0.05). qRT-PCR was performed to further test the effect of PTHrP on the chondrocytes. Biomarkers of proliferation and terminal differentiation, including collagen II, aggrecan, and MMP-13, were investigated. MMP13 is a major enzyme that targets cartilage and leads to the degradation of collagen II and proteoglycan. Aggrecan, a proteoglycan, and collagen II are critical components of the cartilage structure and function of chondrocytes [p<0.01), whilst the level of MMP-13 was lower in the rhPTHrP group than in the control group (p<0.05). In contrast, the level of collagen II increased in the rhPTHrP group  and decreased in the siPTHrP group in comparison with the NC group (p<0.05). The level of aggrecan was also higher in the rhPTHrP group than in the NC group (p<0.05) and lower in the siPTHrP group than in the NC group (p<0.05).To determine the role played by PTHrP in promoting proliferation and preventing terminal differentiation in human knee OA chondrocytes, cells were cultured with siPTHrP, the corresponding controls, or recombinant human PTHrP (rhPTHrP). As shown in drocytes . As showAlizarin Red S staining demonstrated the presence of calcium deposits in the OA chondrocytes. As shown in p<0.01, versus the NC group). Furthermore, the protein levels of PTHrP were analysed by western blotting, which confirmed that miR-15a-5p mimics downregulated the PTHrP protein level (PTHrP 3′UTR contains miR-15a-5p binding sites . To confin level . These rp<0.05) (p<0.01)  . A close(p<0.01) .To understand whether miR-15a-5p has a negative effect on chondrocytes, as shown in p<0.05) (p<0.01).qRT-PCR was performed to further detect the effects of miR-15a-5p mimics on the proliferation and terminal differentiation of chondrocytes. Chondrocytes were transfected with miR-15a-5p mimics and the inhibitors, and the markers of proliferation and terminal differentiation were investigated, including type II collagen, aggrecan, and MMP-13. Transfection with the miR-15a-5p mimics led to decreases in the expression of collagen II and aggrecan (p<0.05) . At the Alizarin Red S (ARS) staining at 12 days was also used to identify calcium deposits in the chondrocytes. As shown in This study reveals a new mechanism of action of PTHrP in the development of OA. We found that PTHrP was upregulated in OA chondrocytes, which is in agreement with several studies in which the expression of PTHrP in OA cartilage was shown to be higher than in normal human articular cartilage , 28. SevAlthough miRNAs play an important role in the regulation and maintenance of normal physiological conditions, in pathological situations, their levels may change due to dysfunction , 32–34. The levels of collagen II, aggrecan, and MMP-13 in chondrocytes have been found to be dysregulated in degenerative joint diseases including OA, and these abnormal expression patterns can therefore be regarded as biomarkers of chondrocyte function , 38. MMP in vitro and will be validated in an animal model in subsequent experiments.Calcium deposition is a marker of chondrocyte terminal differentiation and pathological calcification in osteoarthritic joints , 44. TheWe demonstrated that miR-15a-5p is downregulated in OA chondrocytes in relation to the upregulated expression of PTHrP. MiR-15a-5p promotes the degeneration of chondrocytes by targeting PTHrP. Our findings provide new evidence that may be useful for future research on targeting miR-15a-5p in the treatment of OA.